ALAN DURRANTDepartment
of Agricultural Botany. University College of Wales, Aberystwyth

Received
10.vii.61

1.
INTRODUCTION

IT is
a matter of common observation that responses to nutritional and climatic
differences are not as a general rule transmitted from one generation to the
next. The most that can be said is that, first, possibly some unique situation
might arise involving a particular genotype and a particular environment where
such a heritable change does occur, but of itself having no general
application. Second, all observations may have been made on material suitable
for the investigators, but not for demonstrating the heritable changes. The
most convenient materials are inbred, homozygous strains, which are likely to
be varieties of crop plants bred for high yield in good cultural conditions and
fertile soils. These have been selected for genetic responses to good
environmental conditions to give a phenotype which is economically useful, and
are not representative of naturally occurring organisms. Third, it has received
insufficient attention in biological investigations.

There
is ample evidence, apart from mutation, of the effectiveness of the environment
at all levels in inducing specific types of heritable change. These may be
enumerated as follows; (i) Induced quantitative changes in cytoplasmic
particles (e.g. Sonneborn, 1950). (ii) Enzyme induction in micro-organisms
(e.g. Spiegelman and Campbell, 1956). (iii) Cytoplasmic incorporation of
proviruses (e.g. Malogolowkin, Poulson and Wright, 1959). (iv) Phage
transduction (e.g. Hartman, 1957). (v) Bacterial transformation (e.g. Avery,
MacLeod and McCarty, 1944). (vi) Differentiation of multicellular organisms (e.g.
Waddington, 1956). (vii) Paramutation (e.g. Brink, 1956; Brink, Blackwood and
Notani, 1960; Schaik and Brink, 1959). On these grounds there is no reason to
believe that environmentally induced heritable changes of the more general type
cannot occur, but if organisms can be effectively changed in one generation by
prevailing nutritional and climatic factors, so they should also in the next,
and no heritable change would be observed. On the other hand, should there be
genetic variability among organisms in their ability to maintain, or return to,
a metabolic state that allows response to environmental conditions in
successive generations, then heritable changes will be seen to occur in some
organisms in some environments.

Extensive
experiments have been tarried out during the past seven years, mainly with a
single variety of flax (Linum usitatissimum), on the transmission of
responses to fertiliser treatments applied in one generation,
to subsequent generations. Large heritable changes have been found to occur.
The experiments began in 1953 with studies on the response of several varieties
of flax and linseed, and their F1s and F2s, to the application of all
combinations of nitrogen, potassium, phosphorus and calcium, the object being
to obtain information on their homeostatic properties, gene/environment
interaction, gene interaction and dominance, etc., for a number of characters
in the different environments. Flax and linseed were used because of the
interesting comparisons likely to be given by two distinct types of the one
species, Linum usitatissimum, bred for different characteristics. For several
reasons, some experiments were also tarried out to determine whether any
variability was transmitted from the parents:

First,
it was necessary that this should be checked in view of the type of experiment
envisaged. Second, in other work in the department on inbred lines of Drosophila
melanogaster where the flies had been sib-mated for many hundreds of generations, a large
part of the variability in sternopleural chaeta number was due to the age of
the female parent (Durrant, 1955a), and the chaeta number of the offspring
could also be altered by varying the amount of food and rate of egg laying of
the female parent—changes which are probably either cytoplasmic or maternal
(Durrant, 1955b). Third, flax, unlike linseed and other crop plants, has not
been bred for seed yield, plant weight or plant size but for a relatively long,
unbranched stem with few seeds, and normally grown with as much as one plant to
the square inch in a well established crop. Flax plants might therefore be
expected to be less stable than other crop plants. Fourth, special references
have been made in the literature to flax seed. Percival (1935) states, for
example,

"The
best yield of flax, so far as fibre is concerned, is said by some to be
obtained from seed which has been carefully dried and kept in tightly closed
barrels which exclude moisture for two or three years, experiments having shown
that seed stored in this way gives longer stems and finer bast than fresh seed;
others consider that the highest yield of fibre is secured from the fully
ripened seed, harvested from a crop raised from 'barrel' flax."

Greater
importance is attached to the weight of flax seed than with most other crops,
seed weighing less than 4 grams per 1000 being considered to give a poor flax
crop.

In a
preliminary study, seed was collected from branches which had been pruned to
leave only two capsules per branch to develop, and also from unpruned branches,
of 10 flax varieties, and sown the following year. At maturity the plants from
seed from the pruned branches showed an increase in height of 10 per cent. over
plants from seed from the unpruned branches. Two linseed varieties which were
similarly treated gave no significant response. Another experiment was
therefore begun, and run concurrently with the main experiment, where seed of
one of the flax varieties was collected from plants growing in all combinations
of nitrogen, phosphorus and potassium fertiliser treatments, and sown the
following year. The progeny showed large differences in weight, a result which
initiated the further studies which are to be described.

2.
SYMBOLS

Upper-case
letters are used in making general reference to the fertilisers applied in
these experiments, and also to their main effects and interactions, namely N
(sulphate of ammonia), P (granular triple superphosphate), K (potassium
chloride) and G (calcium hydrate, here referred to as a fertiliser). Lower-case
letters, for example, npkg, nkg, n, pk, denote the combinations of fertilisers
applied, and (1) denotes no application.

Plants
receiving the fertilisers are the parental generation, denoted C0,
and the 1st, 2nd, 3rd, etc. generations from them are denoted C1, C2,
C3, etc. Plants are further designated by the treatments applied to
them or their ancestors and, where necessary, by the year in which they were
applied (not the year in which the particular generation was grown as used
previously, Durrant, 1958). For example plants receiving both N and P in 1954
are denoted C054np, the first generation grown the following year, C154np,
and the second generation, C254np.

Fertilisers
applied to the descendants of the parental generation are parental treatments
for further descendants and in this way successive generations accumulate
several designations, as illustrated in table 1. It is more convenient to
classify plants according to their environmental history, and in abbreviating
these designations the C0 designation is omitted, as shown in the
last column of table 1. The environment in which the plants are growing in any
generation under consideration is then stated separately, or, if added to the
environmental history, prefixed by C0, as for example, for the year
1957 in table 1, np, nk, (1), C057npk.

3.
THE ENVIRONMENT

The
seed was sown in boxes in a warm greenhouse where they received solutions of
commercial fertilisers and the young plants were transplanted into field plots
which had received the same commercial fertilisers as the plants had received
in the boxes. Each individual was traced from the seed to the mature plant so
that selection could not occur unnoticed and seed contamination was prevented.

The
field belonged to the college farm up to and including 1953. It was grazing
land in 1949, 1950 and 1951, cropped with a feeding mixture in 1952 and with
oats undersown with a grass/clover mixture in 1953. In 1952 and 1953 it
received a total of 4 cwt. of compound fertiliser, 5 cwt. of basic slag and 30
cwt. of ground limestone, per acre. The soil is a light to medium loam and,
characteristic of the soils in the area, has a low available phosphate content.

The
fertilisers were sulphate of ammonia (N), muriate of potash (K), granular
triple superphosphate (P) and lime, applied as calcium hydrate (G). The sixteen
combinations of N, P, K and G each at two levels, applied and not applied, were
laid out in the field in 1954 and established as permanent plots. In 1954 each
fertiliser was applied to the appropriate plots at the rate of 6 cwt. per acre,
except for lime which was applied at the rate of 15 cwt. per acre. The npkg
plots therefore had 15 cwt. per acre of lime and a total of 18 cwt. per acre of
the remaining fertilisers. In subsequent years the same quantity of fertilisers
was applied to the plots each year.

The
greenhouse compost was made from 7 parts soil from the field, 3 parts Irish
granulated moss peat and a parts granite chippings. The solutions were made by
dissolving 15 grams each of N, P and K in a litre of water according to the
combination required so that npk, for example, was applied as a 4.5 per cent.
solution. To obtain the phosphate in solution the granular triple
superphosphate was left in water for 24 hours, shaken at intervals and the
solution syphoned off. Two hundred and fifty c.c. of the appropriate solution
was applied at sowing to each box measuring approximately 9 X 14 X 4 in. deep.
The same quantity was applied to days later but using solutions one-sixth the
strength. After each application the boxes were lightly rinsed with water.
Where lime was required ground limestone was mixed with the compost at the rate
of one ounce to the cwt. The plants remained in the warm greenhouse for two to
five weeks depending on the season and space available, placed in a cool
greenhouse for one to two weeks and left outside until they were transplanted
between the ages of four and seven weeks. Just before transplanting, the soil
in the boxes was rinsed with a 0.1 per cent. solution of Gammelin C.L., this
being an effective control of wireworm which would otherwise have caused large
losses in the field. For some of the later experiments it was sufficient to
grow all the plants under uniform, reasonably fertile soil conditions and for
this purpose a compound fertiliser and lime were applied to part of the field,
and John Innes base fertiliser and lime added to the compost used in the
greenhouse.

The
fertilisers can be applied with a fair degree of accuracy but it is of course
impossible to ensure the same environmental conditions each year. In the first
place, the general fertility of the field, maintained at a reasonable level
prior to its use for these experiments, decreased year by year so that
fertilisers which had little or no effect in the first year, had a pronounced effect
in later years. Seasonal variations produced large environmental differences,
drought at transplanting time being a particularly potent factor. Severe
drought at transplanting time, lasting perhaps for six weeks, caused
considerable losses in some experiments and these had to be written off, and
plants in experiments without losses have a vastly different environment from
plants transplanted in a wet, early summer. Sowing of seed generally commenced
at the end of March but continued for as much as a month afterwards, mainly
because of limitations of time and space, and transplanting was also spaced out
over a similar period of time. This time-difference again results in year to
year environmental differences and is the principal reason why all the plants
in some experiments survive during drought, while the loss in others renders
those experiments virtually useless. The seasons interact with the fertiliser
treatments, the more so because of the large differences in size and vigour of
the plants due to the fertilisers applied to the boxes, only some of the plants
being ideally suited for transplanting at any one time, the others being too
large or too small. The use of pot cultures would have removed some of these
difficulties but they would also have limited the experiments because, apart
from the more artificial environment they provide, it would have been possible
to grow only a small fraction of the number of plants each year that were
grown, and the seasonal effects themselves supply additional information—if
these can be correctly construed. It will become evident that it is necessary
for the progeny of plants treated also to be grown under a range of treatments
and if put cultures were used for the parental treatments and larger numbers of
plants of the first generation were planted into the field the following year
there would arise the further problem of relating the two sets of treatments.
With the results of these preliminary experiments now to hand put cultures
would undoubtedly be of more use.

The
environments applied to the plants are, in effect, as much applications of
convenience as they are purposeful. They differ from those normally experienced
by growing erupt in the large amounts of fertilisers applied, the spacing of
the plants so the field at 12-inch intervals in rows 24 inches apart, and in
the higher temperature, and probably higher humidity, they receive its the
greenhouse during the first few weeks of their growth.

4.
THE INDUCTION OF HERITABLE CHANGE

Experiment
1—(i) Origin of the seed and the parental generation

Seed
of the variety Stormont Cirrus was kindly supplied by the Plant Breeding
Station at Stormont in Northern Ireland. In 1953 it was sown in boxes outside
the greenhouse and the plants transplanted (along with other varieties) into
small observation plots which had not recently received fertilisers. The boxes
contained the same soil, which was not made up into compost, as in the
observation plots and no fertiliser was applied to the boxes or to the plots.

Flower
buds were bagged on several plants and seed from eight capsules, four from each
of two plants, used for experiments which began in 1954.

In
1954 five seeds were sown in each of eight boxes to which were added the eight
combinations of N, P and K. No lime was added. The plants were transplanted
into the eight respective fertiliser treatments in the field and at maturity
seed was collected from the five plants in each treatment. The flower buds had
previously been bagged and individual plants within each plot contributed
approximately equal amounts of seed. Seed from this parental generation, C054,
therefore consisted of eight types, npk, np, nk, n, pk, p, k and (1).

(ii)
The first generation

The
first generation, C154, was grown the following year in all combinations of N,
P, K and G. There were sixteen replications of the eight 1st generation types,
two boxes per replication and four rows of six seeds per box. The eight seed
samples, C154, were assigned at random to the eight rows in each pair of boxes
and the sixteen C055 combinations of N, P, K and G assigned at random to the
sixteen replications, giving 128 combinations of C154 and C055 altogether. Out
of a total of 768 seed, 10 failed to germinate and two seedlings were either
abnormal or damaged. Four of those which failed to germinate were in the C154nk
seed sample, the others scattered over the other C154 samples. Five plants from
each row were transplanted into the field, the sixth having been sown as
insurance against poor germination or damage. In the field the eight C154 types
were randomised in each of the sixteen combinations of fertiliser treatments.
These sixteen C055 combinations of treatments applied to the C154 plants may be
regarded as another set of parental treatments for subsequent generations of
plants.

A
sample of ripe seed was collected from each of the 128 plots, all five plants
in each plot contributing approximately equally to each sample as before, but
the flower buds were not bagged. The plants were cut at ground level and
weighed and the mean fresh weight of the five plants in each row is given in
table 2 where the transmitted effects, C154, and the direct effects, C055, of
the fertilisers can be compared in the same group of plants. The analysis in
table 3 shows that there are highly significant differences in both sets. The
C055 treatment totals are less sensitively tested than the C154 totals due to
the split-plot design of the experiment, but among the main effects in both
cases, phosphorus, C055P and C154P, has the greatest overall effect. Summing
over the two levels of calcium and comparing the two sets of totals (table 2,
column totals and row totals B) the npk plants are approximately three times
the weight of the nk plants in both cases. Most of the C154 interactions are
also highly significant, indicating that the balance of fertiliser, applied to
the parents is important. Inspection of fig. 1 shows that C154 and C055(B)
totals are fairly well correlated except for n, the departure of this treatment
being mainly responsible for the non-significant correlation coefficient of
0.52. There is no doubt that there are real differences between the C154 totals
comparable in magnitude to the differences produced by the direct application
of fertilisers, C055.

Table
3 shows that there is evidence of interaction between the plant weights of C154
and C055. To study this further, table 2 may be likened, after summing over the
two levels of calcium, to a diallel table of crosses where the eight
combinations of fertilisers are the eight genotypes, and C154 and C055 the male
and female arrays respectively. The "homozygotes", or
"selfs", are those plants growing in the same environment as their
parents, and the "heterozygotes", or "F1s", those growing
in different environments from their parents. If the plant weights of the
"homozygotes" are greater, or less, than the
"heterozygotes" then "dominance" acts in a negative, or
positive, direction. Using the method of analysis of diallel tables of Jinks
(1954), the regression of array covariances unto array variances shown in fig.
2 has a slope of 0.97, and
is highly significant. The correlation of Vr +Wr, with
the "homozygotes" is positive (r = 0.85) and highly significant, so
that " dominance", which is not complete, acts mainly in a negative
direction. There is no evidence of any other form of interaction from this
analysis which therefore means that plants on average have a greater weight
when growing in the same environment as their parents. This is not due to
adaptation to the environments, as would have been implied by negative
"heterosis" had this occurred, but to the larger types responding
more than the smaller types to higher soil fertility.

There
are four alternative interpretations of these results.

1.
There may be large residual genetic variability in the variety and chance
assortment of the genetic factors among the groups of parent plants receiving
the different treatments produced large phenotypic differences between the
groups which are roughly correlated with the differences produced by the direct
application of the respective fertilisers. This amount of variability was not
evident in the parents and in the first generation the five plants within each
plot were highly uniform.

2.
Large maternal effects may have been responsible. Judged by its weight, table
4, the seed collected in the damp 1954 season from the treated parents, C054,
was of poor quality, and the seed weights also varied between treatments,
although they were not significantly correlated with the plant weights of the
progeny. By comparison, seed collected in the sunny 1955 season from the 1st
generation plants, C154 and C055 combinations, was heavier and more uniform
over the treatments, as shown by the overall means in table 4.

3.
The treatments may have had a selective action on residual genetic variability
in the variety, the selection occurring at sporogenesis, fertilisation or seed
setting, for genetic factors determining roughly the same plant weights as
those produced by the respective treatments. To account for this, some precise,
remarkable, and probably unconvincing mechanism would have to be postulated,
as, for example, only those pollen grains carrying genetic factors for small
plants survive, make faster growth down the style, or take part in fertilisation,
in plants receiving nk.

4.
The parental treatments may have induced heritable changes in the nucleus,
cytoplasm, or in both. The 1st and 2nd interpretations can be separated out by
further experimentation, but the 3rd and 4th are less easily distinguished. At
the sub-cellular level, selection for cytoplasmic particles, or an alteration
in those particles, at any time during the parental generation would have the
same net effect, for both would result in an induced heritable change.

Before
passing on to the second generation some other data recorded will be briefly
mentioned, namely, flowering time, and growth in height, primarily for the
purpose of comparing the effects of the two sets of treatments, C154 and C055.
Flowering time was measured by the number of days after the 30th June when the
first flower opened on each plant. The mean flowering times per plant for C154,
after summation over the C055 treatments, and the mean flowering times for C055
after summation over C154, are given in table 5. There is an overall difference
of 16 days between the C055 treatments but only 3 days between the C154
treatments. The correlation between the two sets is negative (r = -0.3) and
non-significant. Evidently there is no direct relationship between the two sets
which not only makes it less likely that the first generation differences are
due to the carry over of nutrients in the seed but also improbable that any
differences occurring in the second generation (to be described) are due to
transmission of hormones associated with the differences in flowering time of
the first generation plants, since these differences are so small.

The
heights of the plants were measured four times during growth and deviations due
to the overall effects of the applications of P from the mean growth curve of
all 128 plants, calculated for C154 and C055 separately, are shown in fig. 3.
Deviations for plants out receiving phosphorus give mirror images on the other
side of the X axis and are omitted. The deviation due to C154P is small, and the
curve is inverted compared with that of C055P, giving additional evidence for a
real difference in the growth characteristics of the two sets.

(iii)
The second generation-study of the progeny of C154nk and C154npk in 1956

Seed
taken from the first generation plants in 1955 (table s) was used for studies
on the second generation in 1956. The purpose of the first to be described was
to determine whether the difference between two extreme types in the first
generation, C154npk and C154nk, reappeared in the second generation, C254npk
and C254nk, and/or whether the two types responded to the 16 combinations of
treatments in which they were grown in 1955 (C055) to produce a further range
of types in the following generation (C155). The 32 combinations (two C254 x
sixteen C155) were grown in boxes and field plots receiving eight combinations
of treatments (C056), npkg, npk, npg, np, pkg, pk, pg, p, replicated twice. The
4th order interaction, C254/C155N/P/K/G, was confounded between the
replications of the C056 box and field treatments so that there were only 16 of
the 32 combinations of C254 and C155 in any one field plot, and in any one
group of boxes in each replicate receiving the same C056 treatment. As before
there were five plants in each row, giving 1280 plants in all.

The
mean weights of the plants, after summing over the eight C056 treatments, the
two replications and the two levels of lime of C155, are given in table 6.
C254npk is about three times greater than C254nk for all C155npk... (1) giving
no evidence that the difference between C154nk and C154npk has diminished in
the 2nd generation, even though the seed collected from the 1st generation
plants in 1955 was uniform and of good quality (table 4).

In
contrast, the transmitted effects of the fertilisers C055npk... (1), which were
applied in 1955 to the two extreme types, C154npk and C154nk, of the 1st
generation plants of the 1954 treatments, to the 1st generation, C155npk...
(1), are small. The extreme types were analysed separately because they had
different error variabilities. The C155 means within C254npk were not
significantly different. The analysis of the C155 within C254nk is given in
table 7 where the data has been split down sufficient to show that highly
significant differences occur, presumably due to the transmitted effects of the
1955 treatments where, as before, phosphorus has the greatest overall effect.
On the other hand, the correlation of the C155 means within C254nk with the
C055 treatment totals summed over all the plants (table 2, totals B) is low (r
= 0.36), and practically zero with their parental weights (table 2, nk column).
The results of the 1954 treatments are not repeated for, unlike these, the
large differences produced by the 1955 treatments do not reappear in the next
generation and such differences that do appear, 45 per cent. at the most, could
reasonably be ascribed to maternal effects.

(iv)
The second generation-study of all progeny in 1956

In
1956, the progeny of all 128 combinations of C154 and C055 grown in 1955 (see
table a) were replicated four times in npkg plots.

Losses
were too large in the first two replicates, due to drought at transplanting, to
warrant cutting and weighing. There were fewer losses in the remaining two
replicates, the transplanting of which had been postponed, but sufficient to
make detailed analysis not worth while, so that means only are shown in fig. 4
where C254 mean plant weights obtained after summing over C155 are plotted
against the mean plant weights obtained in 1955 for C155 after summing over
C055. This figure, comparing the 1st and 2nd generations of the C054
treatments, shows that the correlation between them is virtually complete (r =
0.97) and the differences have not diminished in the second generation. This result
cannot be due to selection occurring in this C2 where there were
large losses, for the same result was given by the extreme types, npk and nk,
in the previously described C2 plants where no losses occurred. C155
mean plant weights obtained after summing over C254 differed by small amounts,
but gave little indication of any transmitted response from the C055 plants.

These
two 1956 2nd generation studies show that differences similar to those in the
first generation of the 1954 treatments were not obtained in the first
generation of the 1955 treatments, yet the first generation differences of the
1954 treatments are transmitted undiminished to the second generation.
Therefore the most plausible explanation of the differences in the first and
second generations of the 1954 treatments is chance assortment of genetic
factors and not environmentally induced heritable change, nor maternal
inheritance, despite the magnitude of the differences, the uniformity of the
plants and a suggestive correlation between the direct and transmitted
responses to the fertiliser treatments. On the other hand, this may be an
unwarranted conclusion because in the first place the treatments were applied,
and the first generations were grown, its different seasons, and seasonal factors
may be as important as nutritional factors. Secondly, if the 1954 treatments
had induced heritable changes then the plants which received the 1955
treatments must be "genetically" and physiologically different from
those which received the 1956 treatments and they need not necessarily respond
in the same way.

In
1956, seed was taken from the same sample used for the 1954 treatments and 10
plants were grown in each of the eight fertiliser treatments, npkg, npk, nkg,
nk, pg, p, g (1). Table 8 shows that there are large differences in the first
generation which are similar, although not identical, with the first generation
of the respective 1954 treatments. Phosphorus applied by itself now has the
same effect as nk, producing a small type of plant, and only when lime is
applied with phosphorus, and to a certain extent with npk, is the large type
produced in the next generation. Table 8 also shows that the progeny are not
necessarily correlated in weight with their parents, p giving a large parent
plant but a small 1st generation plant, and in the three cases where the second
generation was grown the effects are transmitted to this generation as well.
The discrepancies between these results and those obtained following the 1954,
treatments are probably due to the overall decrease in soil fertility,
particularly in nitrogen and lime.

This
experiment demonstrates that differences are again obtained in the first and
second generations when the original stock of seed is used. Larger and more
profitably designed experiments will be described later which will demonstrate
that the failure of the 1955 treatments
to produce differences in the first generation was primarily due to the type of
plant used, although other factors play a part. It will be convenient to
consider first some characteristics of two extreme types, npk and nk, noting
from the foregoing that their origin cannot be reasonably ascribed to chance
assortment of genetic factors, nor to maternal inheritance.

5.
CHARACTERISTICS OF TWO EXTREME TYPES

Experiment
3—Stability

The
progeny of two extreme types, npk and nk, arising from the 1954 fertiliser
applications were grown for several generations in soil to which had been
applied lime and N, P and K or a compound fertiliser. Table 9 shows that the
npk plants remained about three times the weight of the nk plants, and up to
the 6th generation there is no sign of this difference diminishing. The two
types have been grown in other fertilisers with the same result. Attempts to
reverse them by growing the npk plants in nk and the nk plants in npk produced
only small, and somewhat erratic, deviations in the following generation
similar to those already described (see table 6).

Within
the types, npk and nk, the plants are extremely uniform. Table 10 gives
examples taken from three blocks of an experiment containing, among others, npk
and nk plots with eight plants in each plot. As previously mentioned, seed for
sowing is taken from at least five plants and bulked so that there is no
question of the uniformity being due to the establishment of a type from a
single homozygous individual. Plate 1 shows the two extreme types. The
differences are similar to those which would normally, although not
necessarily, be expected from genetic factors determining quantitative
variation. There are no differences in flower colour, leaf shape or other
morphological characters of this type.

Experiment
4—Grafts

Reciprocal
grafts were made between the npk and nk plants and also between npk plants and
between nk plants in 1957. They were 3rd generation plants of the 1954
treatments which had been grown in npkg in the 1st and 2nd generations. For
these experiments they were grown in compost in pots to which was added npkg.
The grafts were made in the following manner. When the plants were about three
weeks old the stock was prepared by removing the upper part of the plant just
above the attachment of the cotyledenous leaves and making a longitudinal slit
about half an inch long in the stem between the cotyledenous leaves, taking
care not to damage these or the small axillary buds. The scion was prepared
from the terminal 1 1/4 inches of the young plant, the lower portion of which
was cut into a wedge shape after the removal of the leaves in this region, and
inserted into the stock. The join was bound with cotton, and surrounded with
moist peat. They were left for two weeks, during which any roots appearing from
the scion were immediately rubbed off, and then the cotton and peat was
removed. The scion of the mature plant consisted solely of the central,
terminal shoot and the stock included the side branches which arose from the
axillary buds of the cotyledenous leaves.

Table
11 shows that the stock has no effect on the scion. The npk scion weighs about
22 grams on both stocks, and the nk scion weighs about 9 grams on both stocks.
The stock itself is affected by the scion but in a direction expected from the
normal physiological response of side branching to terminal shoot growth. The
totals show that the overall plant weight is determined mainly by the stock.

Seed
taken from the stock and scion of 6 plants of each of the four types of graft
was sown the following year in compound fertiliser. Table 12 shows that in all
cases plants are produced showing the same characteristic difference in weight
as occurs between ungrafted npk and nk plants. There are relatively small
deviations, but they are not consistently in directions that would be expected
had the stock influenced the scion, and vice versa. The difference between npk
and nk is greater in the progeny of the grafts than in the grafts them­selves,
which is doe to the mutilation in preparation, and pot culture, of the grafts.
There is no evidence that the factors determining the difference between the
two types are graft transmitted and therefore they cannot be nutritional nor
hormonal.

Experiment
5—Crosses

Crosses
were made between npk plants, between nk plants, and reciprocally between npk
and nk plants, in 1956, 1957 and 1958. The plants crossed were the 2nd, 3rd and
4th generation plants respectively of the 1954 npk and nk treatments which had
been grown in the intervening generations in npkg. Plants used for crossing in
1956 were grown in their respective treatments of npk and nk, and those used
for crossing in 1957 and 1958 were grown with npk compound fertiliser and lime.
In each case the first generation of the crosses was grown the following year
in npk compound fertiliser with lime. The plant weights are given in table 13.

The
reciprocal crosses of npk and nk are virtually identical in weight so the
factors responsible for the difference between npk and nk are transmitted
equally by the male and female gametes and the nucleus may therefore be
involved. If they were solely cytoplasmic and transmitted through the pollen
they would not necessarily give equilinear inheritance.

These
studies on the stability of the two types, npk and nk, and on their graft
progeny and crossing, show that they are quite distinct and they behave in a
manner similar to different genetic types. The experiments give no direct
support to maternal inheritance, but taken by themselves, they could equally be
taken as confirmation of their origin from the assortment of genetic factors as
from environmentally induced heritable change. This will be considered in more
detail below.

6.
DIFFERENTIATION OF PLANTS

Evidence
was presented earlier for supposing that differences in plant weights occurring
among the descendents of plants receiving different fertiliser treatments were
due to the direct effect of the fertilisers. Two extreme types which have
arisen in this manner were quite stable in the different environments in which
they were grown. Confirmatory experiments (experiments 6 to 10, below) will be
described which will also throw further light on the circumstances of the
effectiveness of the fertilisers in producing changes in some cases, but not in
others.

Experiment
6—induction tests on C154 types

The
purpose of this experiment was to establish whether any of the eight C154 types
had the capacity to change, or whether they were all as unresponsive as the two
extremes, C154npk and C154nk, already studied (table 6). Second generation
plants of the eight C054 parental treatments, whose first generation had been
grown in the C055 treatments of npkg, npk, g and (1), were grown in 1957 in
compound fertiliser with lime. These plants are therefore combinations of C254
and C155. There were four replicates each containing four blocks consisting of
the four C155 types sub-divided into eight plots consisting of the eight C254
types. There were five plants per plot giving 640 plants in all.

The
weights of C254 after passage in the previous generation through npkg and npk
taken together, and after passage through g and (1) taken together, are plotted
against the overall weights of C154 (column totals, table 2) in figs. 5A and 5B
respectively. If all eight C154 types are as stable as the two extremes, npk
and nk, then there should be a constant relationship between C154 and C254
irrespective of whether C154 was grown in npkg, npk, g or (1). There is a
reasonable correlation in both figures, confirming the transmission of the C054
treatment effects to the second generation, but in each case there are two
types, p and k, which are mainly responsible for diminishing the fit to a
linear regression line. After passage through npkg and npk in the first
generation, C254p and C254k plants are large, and after passage through g and
(1) they are small, in relation to the others. In the first case they have
approximately the same weight as C254npk and in the second they approach
C254(1) in weight. The conclusion is that C154p and C154k are the only two
types among the C154 set in which heritable changes can be induced. It is
unlikely that these results have arisen by chance for the movements of both p
and k in response to both combined C055 treatments are in directions expected
from the C055 treatments.

Referring
to table 2, the mean of the weights of the two extremes among the C154 types
(column totals, nk = 11.8, n = 38.7 which approximates closely to npk) is
25.25. The values for C154p and C154k are 26.8 and 25.5 respectively. Thus the
two types in which presumed heritable changes have been induced have weights
midway between the extreme types. C154p and C154k will be referred to as
plastic types to distinguish them from the other C154 types which are stable as
far as it has been shown up to the present.

Experiment
7—Induction tests on four C154 types

This
was similar to experiment 6 except that it was designed to study the responses
of four C154 types, npk, nk, p, (1), to four C055 treatments npk, nk, p and (1)
(see table 2). The progeny were grown in 1957 and consisted of the 16
combinations of the four C254 and the four C155 types. The weights given in
table 14 are the totals of two replicates only, one grown in npkg and the other
in pg (losses in other replicates due to drought at transplanting were too
great to warrant their inclusion) so that the experiment is not particularly
sensitive. The differences between the C254 totals are highly significant which
is in agreement with the results of several previously described experiments.
The C155 totals are significant at the 5 per cent, level only and the interactions
between C254 and C155 are not significant. The C155 differences are in the same
direction as those given in table 8, or similar to the C254 totals here. The
biggest differences in C155 occur among the C254p plants. The percentage
increase of C155npk over C155nk for C254p is 340, compared with 50, 40 and 20
for C254npk, C254nk and C254(1) respectively. Therefore C154p was able to
transmit differences to the next generation following the C055 treatments, and
the others hardly at all, in confirmation of Experiment 6.

Experiment
8—Induction tests on two C154 types

Experiments
6 and 7 agreed in showing that of the C154 types, p, but not npk, nk or (1),
was capable of transmitting responses to fertilisers to the next generation.
Experiment 6 showed that C154k also had this property, and this was tested
again, with C154pk for comparison. The progeny were grown in npk compound
fertiliser with lime in 1959, the same C055 treatments being used as in
Experiment 7. C254k and C254pk were placed in separate blocks each consisting
of eight replications containing the four C155 types. The plant weights are
given in table 15. The differences between the C155 weights are highly
significant (P<0.1 per cent.) for C254k, but not significant for C254pk,
confirming the plasticity of C154k and the stability of C154pk. The differences
between the C155 plant weights of C254k are in the same directions as those
between the C155 plant weights of C254p in table 54 and between the appropriate
means in table 8. These are three independent experiments giving results
similar to those of the initial 1954 experiment, except that on average p now
induces a small rather than an intermediate plant.

Experiment
9—Nutritional lines

Sixteen
lines have been maintained by growing plants in the same fertilisers for a
number of years. These are called nutritional lines. They were descended from
the plants which received the eight treatments, but the eight C154 types which
were grown respectively in the eight C055 treatments were split into two, with
lime, and without lime, so that thereon there were nutritional lines of all
sixteen combinations of N, P, K and G.

Seed
collected each year from the nutritional lines was sown in 1959 in npk compound
fertiliser with lime to establish whether any changes had occurred from year to
year. The sixteen nutritional lines from the four years 1951 1956, 1957 and
1958, were grown in six replicates, each divided into two blocks, fertiliser
combinations with lime being placed in one block and combinations without lime
in the other; each block contained eight sub-blocks consisting of the eight
combinations of N, P and K, and each sub-block contained four plots consisting
of the four years. There were five plants in each plot, giving 1,920 plants
altogether.

The
weights of the plants are shown in figs. 6A and 6B, where are also entered the
weights of C154 (from which all the other plants are descended) given by the
column totals in table 2, but scaled to remove the overall difference in plant
weight between the 1955 and 1959 means.

Although
there was no evidence in experiments described above for maternal effects being
responsible for large differences in weight, the occurrence of a certain amount
of maternal influence on the first generation is likely to occur and might be
expected to be more pronounced in an experiment of this type where plants are
maintained in strongly contrasting environments for several generations, and
figs. 6A and 6B are to be viewed with caution. In both figures, nk maintains a
consistently low value throughout; (1) varies more but nevertheless remains
low. At the other extreme, npk, pk and n remain high although they are somewhat
variable. With calcium, p shows a sudden increase which is maintained
thereafter; k declines slowly. Without calcium, p drops and k rises, both
changes being maintained. The extreme types have remained constant while those
of intermediate weight, p and k, have changed, which is in agreement with the
results of the experiments described above. np shows a decline over the years,
both with and without calcium. There was no previous evidence for the
plasticity of np yet its occurrence here need not be regarded as an exception
for C154np has a plant weight near to those of C154p and C154k. Calculation of
the correlation coefficient of the regression coefficients, ignoring sign, of
plant weight on year, with the deviations, ignoring sign, of the C154 types
from the C154 mean, gives a value of r = -0.78 (P = 2 per cent.). That is, the
greater the deviation from the C154 mean the smaller the consistent change in
subsequent years, as concluded above.

Some
counter observations can be made. With calcium, the fluctuations occurring in n
are almost of the same magnitude as the initial rise in p, while both with and
without calcium, op rises first of all so values that might be thought
characteristic of stable types. Maternal effects could be responsible for the
fluctuations, due to the large variation in seasonal conditions over the years
affecting the quality (i.e. seed weight, germination vigour) of the seed, and the
extremely dry conditions of 1959 when the experiment was carried out would
emphasise these effects. The experiment itself is not sensitive in the sense
that reliance is placed upon the basic fertility of the soil to change, and
then only in specific directions, to provide environmental differences from
year to year.

Taking
all factors into consideration, the results on average do support the
conclusion from previous experiments in attributing plasticity to the intermediate
types and stability to the extremes. There is no evidence that the types become
adapted to their individual environments and, apart from the change shown by
np, perhaps the other feature of interest is the difference calcium makes in
the response to phosphorus and potassium.

Experiment
10—Comparison of stable types with plants grows from the stock seed

If
the plastic types have intermediate weights, then planes grown directly from
the original stock seed, which was used in the first place to demonstrate the
changes, should also be intermediate in weight. In 1958 plants from stock seed
were grown in npkg together with npk and nk plants whirls were descended from
C154npk and C154nk respectively and which had been grown subsequently in npkg
for two years. Three similar experiments were carried out in 1960 but grown in
npk, nk and p respectively. The plant weights given in table 16 are in perfect
agreement with expectation throughout.

Summarising,
these five experiments confirm the occurrence of differences in plant size of
descendants of plants receiving fertiliser treatments and give almost
overwhelming evidence for the occurrence, under the conditions in which the
experiments were carried out, of plastic types which are intermediate in size
between the stable types.

7.
DISCUSSION

A
number of experiments have been described demonstrating the heritability of
changes produced by the application of fertiliser treatments to a single
variety of flax. The transmission does not occur every time but the circumstances
can be resolved into a pattern which allows prediction as to whether
transmission will occur or not. The transmitted responses to the fertiliser
treatments are in general in the same direction in all the experiments and such
exceptions that do arise can be accommodated.

Reviewing
the experimental results, in the first experiment all combinations of N, P and
K were used, and a spectrum of plant weights was obtained in the first
generation. The extreme (stable) types bred true but those of intermediate
weight (plastic types) were capable, after treatment with fertilisers, of
producing plants of both high and low weights in the following generation. In
these further experiments on the plastic types, npk, nk, p and (1) fertiliser
combinations were used, and the transmitted responses were in the same
direction as in the first experiment except that p produced a small type of
plant instead of an intermediate type. When these four combinations of
treatments were again applied to plants grown from the original stork seed at
about the same time as these later experiments, small plants were produced by p
here as well, so that the difference in response to phosphorus must have been
due to different environmental conditions occurring at the two times, the most
likely cause being the decline in soil fertility. A study of sixteen
nutritional lines (plants grown in the same environments for several
generations) showed that no adaptation occurred but gave further evidence for
the existence of plastic and stable types. The original stock seed agreed with
these observations in giving rise to plants which were plastic and intermediate
in weight, In crossing and grafting studies the stable types behaved like
orthodox genetic types giving no evidence of maternal or cytoplasmic
inheritance.

Four
alternative hypotheses were put forward earlier to explain the transmission.
These were, (i) maternal inheritance, (ii) chance assortment of genetic
factors, (iii) selection for genetic factors (on the assumption that in these last
two cases there was a large amount of residual genetic variability in the
variety) and, (iv) environmentally induced heritable change. In view of the
repeatability of the results, chance assortment of genetic factors can be
removed from further consideration.

If
maternal inheritance is defined as the direct influence on the next
generation by the transmission from the female parent of nutrients by reason of
seed size, mineral content, etc., then this hypothesis can also be excluded for
three reasons. The differences are maintained for several generations;
reciprocal crosses between the extreme types have the mid-parent value; and the
progeny of reciprocal grafts are unchanged. If we include with maternal
inheritance the transmission by the female parent of different hormones, or
different concentrations of hormones, built up in the parental environments,
which have a direct effect on the next generation, these also should suffer
dilution over several generations and in the reciprocal grafts, and the reciprocal
crosses should be like the female parents. If it be supposed that hormones,
having a direct effect on the next generation, are equally effective when
transmitted through the pollen or through the egg cell, then the reciprocal
crosses would have the mid parent value but dilution effects would again become
apparent.

If
certain nutrients or hormones built op by the parent plant have an indirect
effect then dilution need not necessarily occur. If, for example, a nutrient or
hormone is transmitted equally by the male and female parents and is capable of
differentiating the plant at an early stage, as the seed ripens 00 the parent
plant, or somewhat later, the differentiated plant would have the same
characteristics as its parents and manufacture the same nutrients or hormones,
the early differentiation overriding any influence of the environment in which
the plant is grown. This could continue from generation to generation
indefinitely. Although the reciprocal grafts were made at an early age it could
be assumed that the stock and scion were already differentiated, but further
assumptions would have to be made for the progeny of the stock and scion to
retain their individual characteristics. The hormones or nutrients must remain
almost completely localised, or diffuse very slowly from one so the other, or
act only in those cells which are linearly descended from the early
differentiated tissue which gave rise to the hormone. Differentiation has to be
postulated and once postulated is sufficient in itself to provide an
explanation, and an hormonal interpretation becomes superfluous. Hormonal
differences may well occur between the types but they cannot satisfactorily be
considered to be responsible for the transmission from one generation to the
next, and are more likely, in this context, so be the result, rather than the
cause, of differentiation.

Differentiation
implies heritable change, which leads to the examination of the remaining two
alternative interpretations; selection for genetic factors and induced heritable
change. Assuming large residual genetic variability in the variety, infallible
selection each time by the treatments for chromosomal factors, at some stage
prior to seed formation, is unlikely, although it has not been disproved.
Selection for heritable cytoplasmic particles could have occurred in view of
the known influence of the environment on such particles in unicellular
organisms where the environment may influence differentially the rates of
division of cell and particles—or the particles themselves may have undergone
change. Equilinear inheritance would require that they are transmitted equally
through the pollen and the egg cell, or if fewer in number in the pollen,
divide more rapidly at some subsequent stage. Equilinear inheritance suggests
that the nucleus is implicated, and the nucleus must be taken into
consideration in any case because of the biochemical inter-relationship between
the nucleus and the cytoplasm, and of the influence the environment can have on
the activity of the nucleus through the intermediary of the cytoplasm, as, for
example, in the environmentally induced antigen changes in Paramecium (Beale, 1954).

Where
the chromosomes are homozygous and discounting mutations, possible nuclear
changes may be roughly divided into three, not mutually exclusive, groups: (i)
Structural; for example, multiplication of chromosome threads, quantitative
differences in nucleic acid, amount of coiling in certain regions. None of
these differences has been detected with the light microscope. (ii) Chemical;
either in the nucleoplasm or in the chromosomes. (iii) Functional; the
differential stimulation of chromosomal regions in their participation in
metabolic activities by changes in the cytoplasm or in other parts of the
chromosomes. It would be more reasonable to suppose that nuclear changes
directed by the environment would fall into one or more of these categories
rather than to assume that the environment selects infallibly certain members
of a heterozygous set of chromosomes, or one chromosome of an heterozygous
pair.

The
induced changes in flax resemble differentiation in multicellular organisms.
The intermediate, plastic type (≡ undifferentiated cell) may be changed
to a large stable type or a small stable type (≡ differentiated cells)
depending upon the environment in which it is grown. There is good evidence
that the nucleus is involved in differentiation from nuclear transfer
experiments, both in unicellular organisms (Danielli, Lorch, Ord, Wilson,
1955), and in multicellular organisms (King and Briggs, 1955), which may have
their counterpart in the induced changes in flax, but with the characteristic
difference that these survive meiosis.

Leaving
on one side the question as to whether quantitative, chemical or functional
changes have occurred in self-reproducing particles in the cytoplasm or in the
nucleus, the most useful working hypothesis at this stage with regard to the
changes in flax is that both cytoplasm and nucleus contribute in establishing a
metabolic equilibrium when the plants are grown under certain environmental
conditions which is not disturbed thereafter when the plants are grown within
the range of environments supplied in these experiments. Since the
environmentally induced types parallel in heredity differences due to orthodox
genetic factors, and to separate them from epigenetic changes, they may be
referred to as genotrophs resulting from genotrophic change.

Turning
to the effects of the individual fertilisers in inducing heritable changes,
these need to be interpreted against the background of the inherent fertility
of the soil. In the first experiment (table 2), phosphorus gave the greatest
overall difference in the following generation, which suggests that alterations
in nucleic acid synthesis might be primarily responsible for the inherited
differences. On the other hand, the soil was relatively deficient in
phosphorus, judged by the responses so the direct applications of the
fertilisers, and the application of phosphorus to the parent plants may have
merely allowed them to make better use of the nitrogen and potassium which were
applied, or other minerals in the soil which were not applied. Phosphorus
applied by itself produced plants of only intermediate size in the next
generation, whereas nitrogen by itself produced the largest type of plant even
though when applied directly so the plant it produced a relatively small type
(table 2). Consequently nitrogen and not phosphorus may be primarily
responsible for inducing large plants in the next generation. On the other
hand, nk induces a large plant and pk a small plant, but it is probable that,
judged from the appearance of seedlings, nk had an inhibiting effect on their
growth from which they were not able to recover in the absence of phosphorus,
and plants receiving phosphorus and potassium were able so take up and utilise
the nitrogen which was available in the soil.

Pursuing
this hypothesis, in later years, when there was less nitrogen in the soil,
phosphorus applied alone gave the smallest type of plant, comparable with the
nk type, even though the parent plants receiving the phosphorus were larger
than the plants receiving any of the other treatments, including npk (tables 8,
14, 15). In the experiment assessing the nutritional lines, the np nutritional
line, both with and without calcium, gradually decreased in plant weight (figs.
6A and 6B). Presuming that the np type had retained its plasticity, this would
be due to the gradual loss of nitrogen from the soil so that the initial
combination of nitrogen and phosphorus plus soil nitrogen, inducing a
relatively large type, becomes progressively a combination of nitrogen and
phosphorus with only small amounts of soil nitrogen to induce a relatively
small type. On this assumption, the character of the progeny is dependent upon
the relative concentrations of nitrogen and phosphorus applied to the parents.
Phosphorus without nitrogen produces large parent plants but induces small
plants in the next generation; nitrogen without phosphorus produces small
parent plants but induces large plants in the next generation. When nitrogen is
limiting, and when plants are stimulated to grow rapidly with the application
of phosphorus, perhaps the synthetic sequence, nitrogen—amino-acid—protein
(enzyme), is also limiting. This parallels the situation in bacteria where
enzyme induction only occurs when there is a sufficient supply of nitrogen in
the media. In all ways this may be an over-simplified interpretation.
Clarification is required of the interactions of nitrogen and phosphorus with
calcium and potassium and of other soil nutrients, and of the negative
parent/offspring correlation.

Considering
the general application of these results, in some respects flax is unique among
crop plants. The commercial varieties have not been selected for plant, root,
stem or leaf weight, nor for fruit or seed yield, but for a long stem with few
side branches and little seed production, containing cellulose fibres of
commercial quality, and grown under conditions favouring these characters, that
is, in soils of not high fertility and at seed rates producing on average one
plant to the square inch. These are conditions which might be expected to
produce plants of intermediate size compared, under suitable conditions, with
plants obtained by selecting for large and small plant weights. In these
experiments the plastic type was intermediate in weight compared with the
stable types to which it gave rise. Selection for large plants, or maximum seed
yields, may produce plants containing more stable heritable factors, so that
although these may supply suitable homogeneous and homozygous material they may
be less suitable for this type of investigation. Individuals in a natural
population might be expected to vary in their ability to transmit changes to their
offspring in response to environmental stimuli due to both genetic variability
and to differences in the environmental history of their ancestors.

The
above generalisation is probably only partially correct for the contrast drawn
between flax and other crop plants does not necessarily mean that other crop
plants are incapable of changing. In the first place, although exceptionally
large doses were applied in these experiments, it is possible that at some time
in the environmental history of the ancestors of the stock seed used,
conditions of high or low fertility, or excess of nitrogen or phosphorus, were
encountered which should, according to the above reasoning, have produced
stable types. In the second place, flax seed is not necessarily obtained from a
flax crop, but from specially cultivated plants. Mercer (1948) states,
"First-class fibre and first-class ripe seed may not be produced from the
same crop". A climate suitable for flax growing is not suitable for flax
seed production and as a consequence flax seed has in the past been imported
into Northern Ireland. It is not certain how relevant these observations are to
the present discussion but it is certain that factors other than nutritional
should be taken into consideration, and in particular, temperature.

The
precaution was taken of growing the first generation of plants of the original
stock seed in 1953 in boxes in ordinary soil taken from the field, and left in
the open. Plants of subsequent generations, receiving the fertiliser treatments
were grown for the first few weeks in a heated greenhouse where the temperature
during the day and night was considerably higher than outside, and it is more
than likely that the heritable responses are due to the combined effects of
temperature and nutrition. Exploratory experiments suggest that this is true.
Similarly, the stable types may out be stable when placed in the correct
combination of temperature, nutrition and day length, but revert to the plastic
type. This might give rise to a situation where a sequence of environments is
required to produce the large from the small, so called, stable type. On the
other hand, those plants which have been shown to be extreme types under the
conditions of the experiments described here, do not necessarily differ in
appearance in all environments, although breeding tests demonstrate that they
are different. Further discussion on this topic would be to trespass beyond the
results presented in this paper.

In
discussing the authenticity of the inheritance of acquired characters in higher
organisms writers have stated the paradox inherent in the concept:—if the
environment is capable of changing plants in one generation then it should be
equally capable of changing them again in the next generation, and therefore
there should be no observed inheritance of acquired characters. It becomes more
acceptable when modified to:—changes produced in parent organisms by some
environments may reappear in the offspring when grown in some environments. One
is dealing therefore with the degree of permanency of heritable factors, where
the concept of heritable factors encompasses the frequency, composition,
activity and interaction of nuclear and cytoplasmic particles.

It is
of advantage to the organism for heritable factors to vary in their permanency
for, at one extreme, it is able to retain its overall character, balance and
evolutionary gains, and at the other, to adapt itself rapidly to current
environmental conditions within its lifetime. Temporary differences occurring
between offspring of parents grown in different environments are generally
classified as maternal effects, but in some cases they may be due to heritable
factors differing only from these in the flax experiments in being more
transient. The same may apply to differences between reciprocal crosses,
particularly when these are in opposite directions to the maternal parents, as
occurs in some flax and linseed crosses in certain environments (Durrant,
unpublished). Lints (1960) has also demonstrated this in Drosophila melanogaster. In Drosophila too there may be a balancing,
or compensating, mechanism (Durrant, 1955b, details to be published), which
prevents the organism swinging too far in one direction. Even diurnal
endogenous rhythms in Drosophila parents can be recorded in the offspring (Durrant,
unpublished). This is a marginal region where genetic and maternal
(nutritional) inheritance meet.

Heritable
factors responsible for the differences between the flax types result from the
interactions of the environment with other heritable factors, permanent and
transient, contained in the plant. There are several aspects to the question as
to whether they are permanent in the same way as chromosomal genes. The two
extreme types of flax have remained stable for six generations under the
conditions of these experiments irrespective of the nutritional environment. In
appearance, uniformity, and reciprocal crosses and grafts, they behave as two
distinct genetic types, although it is possible that they would revert if the
right combinations of all the environmental factors were supplied. Presumed
genetic differences between other plants could conceivably have arisen in the
same way.

If
the changes are nuclear, as mentioned above, they may be due to interaction of
the nucleus with the cytoplasm or to alterations in the chemical composition of
the chromosomes or, as discussed by Matter (1961), in nuclear substances
associated with the chromosomes. For the environment to be directly concerned
in evolution other than as a selective agent, changes in the more permanent
chromosomal material would be necessary, either directly, or indirectly by way
of the associated material which is then eventually incorporated into the more
permanent chromosomal structure. On this view, fixation need not be immediate
but related to the time of exposure to the specific treatments. Although there
is no evidence at present, complete reversion of a genotrophic change may not
always be possible. Instead, small amounts of additive and accumulative
variation might be fixed in each generation which would contribute to the
overall genetic variability determining quantitative variation. By analogy,
homeostatic changes in metabolism associated with gene mutations at other parts
of the chromosomes might also be permanently fixed, so that there would arise
several heritable changes at the one time. In any case, the environment would
have an indirect evolutionary effect, for, apart from inducing the genotrophs,
selection by the environment would act differentially on them, and for
mutations occurring within them. On outcrossing the genotrophs, new genetic
constitutions could permanently fix the environmentally induced changes, as
also could the environmental selection for specific genes if the changes had
been induced in heterozygous populations.

It is
difficult to say how far these results are compatible with those described by
eastern geneticists until unequivocal information is available on the details
of their experimentation, and on how much segregation of chromosomal factors
contributed to the differences. The occurrence of plants that may be plastic or
stable depending upon the particular set of environments being investigated,
and upon their ancestral environmental history, could lead to confusion in the
interpretation of the lack of repeatability of results.

As
regards crop improvement, although larger and smaller plants have been obtained
from the original flax variety, neither of these may be better economically
than the original, intermediate type.

The
environments supplied, and experimental procedures adopted were those most
convenient for preliminary experiments. Other experiments which are in progress
fall into four main groups: (1) Critical assessment of the environmental
factors—minerals, temperature, light intensity, day length—concerned in the
induction, and possible reversion, of heritable change. (2) Assessment of
crosses between stable types and between stable and plastic types grown in
different environments to obtain information on the interactions between
environment, cytoplasm and nucleus. (3) Induction of heritable changes in other
species and individuals from natural populations. (4) Biochemical and
physiological studies on the stable and plastic types, and studies on the
effect of growth hormones, their behaviour in different environments and on
crossing with other varieties.

8.
SUMMARY

Under
the conditions detailed, heritable changes have been induced in a flax variety
by growing the parent plants in different combinations of fertiliser treatments.
A plastic type is defined which is changed into a larger form or a smaller
form, depending on the fertiliser applied. Both the large and the small forms
are stable and have remained unchanged for several generations under the
condition of these experiments, irrespective of the fertilisers subsequently
supplied.

The
three types are termed genotrophs. The plastic genotroph, and probably the stable
genotrophs, have these properties only within a particular range of
environments. When the small and large genotrophs are reciprocally grafted and
reciprocally crossed they behave like two distinct genetic types, and the
nucleus is probably involved in the genotrophic change.

Analogies
are drawn with enzyme induction in micro-organisms and differentiation in multicellular
organisms. The balance of nitrogen and phosphorus in the nutrients applied
plays at least some part, if not a major part, in the induction of heritable
change.

The
induction of heritable change is dependent upon the environmental conditions supplied
and the plastic properties of the plant, the plastic properties being dependent
upon its genetic constitution and the environmental history of its ancestors.

Flax
has not been selected for grain yield nor plant weight and it is therefore
likely to be more plastic than other crop plants where selection and
environment may have resulted in the establishment of more stable types, but
certain combinations of environmental factors involving nutrition,
temperatures, day length and light intensity may be effective in inducing
changes in these also. Inbred lines from natural populations would be
favourable material for similar investigations provided no deliberate selection
was employed and each was split into sub-lines kept in different environments
during their establishment.